DTIC ADA533786: Bone Mass Gained in Response to External Loading is Preserved for Several Weeks Following Cessation of Loading in 10 Week C57BL/6J Mice

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J Musculoskelet Neuronal Interact 2010; 10(4):274-280
Original Article Hylonome
Bone mass gained in response to external loading is
preserved for several weeks following cessation of
loading in 10 week C57BL/6J mice
C. Kesavan1,2and S. Mohan1,2
1Musculoskeletal Disease Center, VA Loma Linda Healthcare System, Loma Linda, CA 92357, USA;
2Department of Medicine, Physiology and Biochemistry, Loma Linda University, Loma Linda, CA 92354, USA
Abstract
Objective:Dynamic loads lead to increases in bone mass. How long these gains are maintained after cessation of loading, how-
ever, is not fully understood. Methods:A long term study was performed in which skeletal changes were monitored by pQCT
every 2-4 weeks (wks) for a 12 wk period after application of external loading using four-point bending device on 10 wk old female
C57BL/6J mice. Results:2 wks of loading caused 15-40% increase in bone parameters (vBMD, cross sectional area (CSA)) and
bone strength (yield load, maximum load and toughness). Positive correlations between these two parameters (r= 0.72 to 0.88,
p<0.05) suggest that the changes in bone parameters induced by loading are responsible, in part, for the increase in bone strength.
Once loading is terminated the bone response did not continue. The vBMD gained by loading was significant for a period of 5 wks
and returned to the levels of controls at 12 wks. The CSA though declined but was still significantly elevated at 12 wks. Bone
strength showed no difference between loaded and non-loaded bones at 12 wks. Conclusion:Our results show that external loading
increased bone mass, was maintained for several weeks after termination of last loading.
Keywords:Density, Bone fate, Physical Exercise, Rehabilitation, Mice
Introduction and prevent osteoporotic fractures in men and women.
Clinical studies in young and postmenopausal women sub-
jected to treadmill exercise have shown that exercise induced
Mechanical loading (ML) is an effective stimulator of bone
benefits, such as increases in vBMD and bone mineral content
formation. Past studies using animal and human models have
(BMC), are eventually lost if exercise is ceased completely10.
shown that loading increases bone formation while non-loading,
Similar data was presented by Vuori et al. when reporting that
such as prolonged bed rest, immobilization, and space flight, re-
unilateral leg presses done four times a week, for 12 months in-
sults in the increase of bone loss1-9. We, and others, using inbred
creased bone mass, but returned to pre-training levels with only
strains of mice, have reported that ML causes increases in the 3 months of retirement from exercise11,12. Another study involv-
volumetric bone mineral density (vBMD) when measured by ing gymnasts also showed that bone density gained by long term
peripheral quantitative computed tomography (pQCT)2,3. Thus, loading declines followed by off season (unloading)13. Thus, data
physical exercise has been used as a strategy to maintain BMD from various independent studies in humans suggests that exer-
cise induced bone mass benefits erode over time. Animal studies
using a rat model have shown that increased femoral bone den-
sity, gained through treadmill exercise, resulted in a decreased
The authors have no conflict of interest. bone formation rate after deconditioning14,15. However, the issue
of how deconditioning affects bone mass maintenance in
Corresponding author: Subburaman Mohan, Ph.D., Musculoskeletal Disease
Center (151), Jerry L. Pettis Memorial VA Medical Center, 11201 Benton mechanosensitive mouse model is not well understood.
Street, Loma Linda, CA 92357, USA We, and others, have previously shown that compared to all
E-mail: [email protected] other mouse strain, C57BL/6J (B6), a low bone density mouse,
responds well to ML. We have reported that 2 wks of ML by
Editedby: J. Gasser
Accepted 22 September 2010 four-point bending causes a 10-15% increase in tibia vBMD
274
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Bone mass gained in response to external loading is preserved for several
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weeks following cessation of loading in 10 week C57BL/6J mice
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14. ABSTRACT
Objective: Dynamic loads lead to increases in bone mass. How long these gains are maintained after
cessation of loading, however is not fully understood. Methods: A long term study was performed in which
skeletal changes were monitored by pQCT every 2-4 weeks (wks) for a 12 wk period after application of
external loading using four-point bending device on 10 wk old female C57BL/6J mice. Results: 2 wks of
loading caused 15-40% increase in bone parameters (vBMD, cross sectional area (CSA)) and bone strength
(yield load, maximum load and toughness). Positive correlations between these two parameters (r= 0.72 to
0.88 p<0.05) suggest that the changes in bone parameters induced by loading are responsible, in part, for
the increase in bone strength. Once loading is terminated the bone response did not continue. The vBMD
gained by loading was significant for a period of 5 wks and returned to the levels of controls at 12 wks. The
CSA though declined but was still significantly elevated at 12 wks. Bone strength showed no difference
between loaded and non-loaded bones at 12 wks. Conclusion: Our results show that external loading
increased bone mass, was maintained for several weeks after termination of last loading.
15. SUBJECT TERMS
16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF 18. NUMBER 19a. NAME OF
ABSTRACT OF PAGES RESPONSIBLE PERSON
a. REPORT b. ABSTRACT c. THIS PAGE Same as 7
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C. Kesavan and S. Mohan: BMD fate after cessation of loading
Figure 1.A schematic representation of the study design. (a) In group-I, mice were euthanized on day 14 after in vivopQCT measurement of
bone parameters, followed by tibias were collected and stored at 4ºC for mechanical testing. (b) In group-II, mice were euthanized on day 96
after in vivopQCT measurement of bone parameter, followed by tibias were collected to perform mechanical testing.
and a 30-40% increase in cross-sectional area in 10 week old measurement at the end of loading and at 2, 4, 6, 8 and 12
female B6 mice3. Thus, in this mechanosensitive B6 mouse, weeks after loading. Mice were euthanized at 12 week after in
there is a robust increase in both vBMD and bone size after 2 vivopQCT measurement of bone parameter. Tibias were then
wks of four-point bending, but, how long this newly formed collected to perform mechanical testing. A schematic represen-
bone, induced by four-point bending is maintained after ces- tation of the study design is shown in Figure 1.
sation of loading is still unclear. Furthermore, to our knowl-
edge, no study has examined whether bone mass or bone Peripheral quantitative computed tomography (pQCT)
strength gained by four-point bending is maintained or lost measurements
after termination of loading and hence, the present study was
To measure loading induced changes in bone parameters in
carried out to address the above issue.
the loaded and non-loaded bone, we used pQCT (Stratec XCT
960M, Norland Medical System, Ft. Atkinson, WI) as described
Materials and methods
previously3,16. The resolution of pQCT scan is 70 micron. In vivo
Mice pQCT measurements were performed at immediate (0-time
point), 2, 4, 6, 8, and 12 wks after the last loading regimen.
10 Female C57BL/6J were purchased from the Jackson
Laboratory (Bar Harbor, Me). All mice were housed under Mechanical properties of bone
standard conditions of 14-hour light and 10-hour darkness, and
Tibiae were stored frozen in gauze moistened with PBS and
had free access to food and water. The body weights of these
thawed in PBS at 4°C. The anterior-posterior diameter (AP.Dm)
animals measured before the initial loading were 18.32±0.79
and lateral-medial diameter (LM.Dm) were measured with
grams. The experimental protocols were in compliance with
calipers. The tibiae were tested by three-point bending using
animal welfare regulations and approved by our local IACUC.
the Instron DynaMight testing system (Model 8840; Instron,
In vivo loading model and peripheral quantitative computed Canton, MA, USA) as previously described17. Each tibia was
tomography (pQCT) measurements placed on two immovable supports which were 5 mm apart. An
initial 1.0 N was applied on the tibia at a position of 2.10 mm
At 10 wks of age, two groups (n=5/group) of female B6 mice
away from the tibia-fibular junction to prevent the rotation of
were subjected to ML using the four-point bending device as
described previously3. The loading protocol consists of a 9.0 the bone due to the shape of the tibia and to be consistent in
breaking region between the bones. Furthermore, this area cor-
Newton (N) force (9N produce 3682 micro strain) at a fre-
responds to the 4 mm loading region3,16. It was then loaded from
quency of 2 Hz for 36 cycles, performed daily under inhalable
anesthesia (5% Halothane and 95% oxygen). The loading pro- this midpoint at a constant rate (2 mm/minute) to the point of
cedure was repeated for 6 days/week with 1 day of rest for 2 fracture. Load displacement curves were used to calculate yield
wks. The right tibia was used for loading and the left tibia as load (Py), maximum load (Pmax) and toughness (Ut). Cross sec-
contralateral internal control in each mouse (From onwards in tional moment of inertia was calculated from the measured an-
this manuscript, we will call right tibia as loaded bone and left terior-posterior diameter and lateral-medial diameter and from
tibia as non-loaded bone). In group-I, mice were euthanized by the average cortical thickness obtained by pQCT analysis as
carbon dioxide inhalation on day 14 after in vivopQCT meas- previously described18,19. The number of mice used for this ex-
urement of bone parameters. Tibias were collected and stored periment is n=5 of which one mice had a technical problem in
at 4ºC for mechanical testing. In group-II, mice were subjected breaking the bone, and was excluded from the study. Thus, data
to 2 weeks of mechanical loading followed by in vivo pQCT from only 4 mice were used for this analysis.
275
C. Kesavan and S. Mohan: BMD fate after cessation of loading
Statistical analysis
Data are presented as Mean ± Standard error (SE). Loading
induced changes in skeletal parameters were determined by cal-
culating percent changes in the loaded versus non-loaded bones
of the same animal to avoid any inherent variation between an-
imals. One-way ANOVA (Newman-Keuls Post Hoc test) was
used to evaluate the influence of time on the loaded and non-
loaded bones. Correlation matrices (values for bone parameters
and mechanical properties of respective mouse were used for
correlation) were used to determine whether there is any asso-
ciation between changes in skeletal parameters and mechanical
properties. Standard t-test was used to compare skeletal changes
between loaded and non-loaded bone. We used STATISTICA
software (StatSoft, Inc version 7.1, 2005) for our analysis and
the results were considered significantly different at p<0.05.
Results
Bone anabolic response after cessation of loading
Two wks of four-point bending increased bone mineral con-
tent (BMC) by 45% in the loaded bone compared to correspon-
ding non-loaded bone. The increased BMC was maintained
(10-45%) throughout the 12 week cessation period after last
loading (Figure 2a). The increased BMC in the loaded bone is
caused by both bone size and vBMD changes. Bone size, as
reflected by periosteal circumference (PC), was increased by
18% after two wks of ML and was significantly different from
corresponding non-loaded bone throughout the entire study
(Figure 2b). vBMD was increased by 14% after two wks of
ML and remained high compared to non-loaded bones only
until 4 wks after cessation of exercise (Figure 2c).
ANOVA analysis revealed that absolute values of non-loaded
bones for PC were not different between the post loading time
points while in the loaded bones, PC values of 6, 8 and 12 wks
were different (p=0.03) from 0, 2 and 4 wks post loading time
points (Figure 2b). Similarly, for vBMD, the absolute values of
loaded bones showed no significant different between the post
loading time points while the absolute values of non-loaded
bones at 6, 8 and 12 week were significantly different (p<0.05)
from 0, 2 and 4 week post loading time points as evident from
ANOVA analysis (Figure 2c). In the case of BMC, the values
of loaded bone were not different between the post loading time
points while in the non-loaded bones, the values of 12 week
were significantly different from 0, 2, 4, 6 and 8 wks of post
loading time points (ANOVA analysis). Figure 2.Changes in bone parameters measured at different time
Figure 3 shows percent changes in loading induced in- points after cessation of loading. The y-axis corresponds to absolute
creases in cross sectional area (bone size), cortical area, corti- changes in bone parameters in response to 12 days four-point bending
cal thickness and vBMD as a function of time after cessation on 10 week female C57BL/6J mice. The x-axis corresponds to various
time points. (a) Bone mineral content (BMC), (b) Periosteal circum-
of last loading. Mechanical loading induced gains in all four
ference (PC) and (c) total volumetric Bone mineral Density (vBMD).
parameters declined with time but at different rates. The
Values are mean ± SE, ap<0.05 vs. non-loaded bone, N=5. #PC loaded
vBMD and cortical thickness gained after two weeks of load-
values of 6-, 8- and 12-week are different from 0-, 2-, and 4-week
ing in the loaded bone was significant over non-loaded bones
(p<0.05, ANOVA, Newman-Keuls Post Hoc Test). ҰvBMD non-
for a period of 5-6 weeks and then returned to the levels of loadedvalues of 6-, 8- and 12- week are different from 0-, 2-, and 4-
control at 12 weeks. However, the gain in the CSA and cortical weeks (p<0.05, ANOVA, Newman-Keuls Post Hoc Test). $BMC non-
area after two weeks of loading though declined but still was loadedvalues of 12 week are different from 0-, 2-, 4-, 6- and 8-weeks
significantly elevated at 12 weeks. (p<0.05, ANOVA, Newman-Keuls Post Hoc Test).
276
C. Kesavan and S. Mohan: BMD fate after cessation of loading
Figure 3.Changes in CSA and vBMD measured at different time points after cessation of loading. The y-axis corresponds to percent change
and x-axis corresponds to duration post-loading (weeks). CSA, Cross sectional area and vBMD, total volumetric bone mineral density, values
are mean ± SE, ap<0.05 vs. non-loadedbone, N=5.
Parameters 0 week 12 week
Non-loaded bone Loaded bone Non-loaded bone Loaded bone
Yield load (N) 14.48±0.42 20.34±0.84* 17.64±0.72 16.44±0.13
Maximum load (N) 18.54±0.67 24.05±0.58* 21.93±0.39 21.05±0.17
Toughness (N/mm2) 2.17±0.03 3.30±0.10* 2.03±0.02 1.94±0.14
Abbreviations: N, Newton
Values are mean ± SE with four mice per group, *p<0.05 vs. non-loaded bones.
0- and 12-week mechanical parameters of loaded bonesare significantly different (ANOVA, Newman-Keuls Post Hoc test).
0- and 12-week mechanical parameters of non-loaded bonesare not significantly different, p=0.07 (ANOVA, Newman-Keuls Post Hoc test).
Table 1.Changes in mechanical properties of bone measured at 0- and12-weeks after the cessation of last loading.
Bone mechanical properties after termination of loading However, at 12 wks, there was no difference in the above pa-
rameters between loaded and non-loaded bones (Table 1). Fur-
In order to determine if the increase in skeletal parameters
thermore, we also performed a correlation analysis between
reflects an increase in bone mechanical properties, we per-
these mechanical properties vs. vBMD, CSA and cortical
formed a bone breaking study on the loaded and non-loaded
thickness to elucidate whether increase in bone mechanical
bones of mice immediately after the loading and at 12 wks
properties is due to an increase in the changes in bone param-
after cessation of loading. We found that immediately after 2
eters, measured by pQCT. The results show a significant cor-
wks of loading, yield load, maximum load and toughness were
relation between vBMD, area and cortical thickness with yield
significantly increased by 42%, 29% and 42%, respectively in
load, maximum load and toughness (r= 0.72 to 0.88, p<0.05).
the loaded bones compared to non-loaded bones (Table 1).
277
C. Kesavan and S. Mohan: BMD fate after cessation of loading
Discussion
In this study, the choice of inbred strain of mice, the loading
device and regimen used were based on previous findings3,16.
Two wks of four-point bending on the right tibiae when com-
pared to left tibiae resulted in a significant increase in bone
parameters, such as BMC, CSA and vBMD, as shown in Fig-
ure 1. The magnitude of increase in skeletal parameters after
2 wks of loading is consistent with our previous study3. How-
ever, in this study, we show that this increase in CSA (40%)
and vBMD (15%) gained immediately after 2 wks of loading,
did not continue over time after the cessation of last external
loading. The decrease in the difference in CSA between non-
loaded and loaded bones over time raises a question of whether
Figure 4.Cross sectional area of non-loadedvs. loaded bone. This
this is due to loss of bone mass acquired during loading or due
figure shows the newly formed bone at the periosteal site in response
to the influence of growth in the loaded or non-loaded bones. to 2 weeks of loading and its fate over time after cessation of loading.
The findings from our study illustrate that the reduction in The arrow corresponds to newly formed bone and the white color rep-
CSA difference between non-loaded and loaded bones over resents cortical bone.
time was purely due to a gradual loss in gained bone and not
due to growth as evident from ANOVA analysis. In the case
of vBMD, we found a significant increase in vBMD with ad-
vancing age (i.e. between 12 and 22 wks) by ANOVA in the maintained for several wks after the cessation of the last external
non-loaded but not in the loaded bone. It is possible that loading and this is consistent with the earlier reports in rat and
vBMD increased in the non-loaded bone is to compensate for human model20-22.
the increased body weight during this period while in the Since in our study, we found a significant increase in bone
loaded bones this did not occur as the vBMD is sufficiently parameters in response to loading and because earlier reports
increased due to the four-point bending. It remains to be de- have shown that an increase in bone parameters leads to an in-
termined whether the lack of vBMD difference between the crease in bone mechanical properties, we anticipate a measur-
loaded and non-loaded bones at 12 wks after cessation of ex- able difference in mechanical properties between the bones
ternal loading is due to a difference in bone accretion rates be- immediately after loading and at 12 wks. Accordingly, the
tween non-loaded and loaded bones between 12 and 22 wks findings from our study revealed that bone strength was sig-
of age and/or due to partial loss of ML-induced newly formed nificantly increased by 29 to 42% immediately after 2 wks of
bone after cessation of loading in the loaded tibia. loading (Table 1) and this increase in bone strength is in part,
In the pQCT analysis of bone parameters using the lower mediated by an increase in vBMD and CSA as evident from
threshold (180-730), we found that the magnitude of increase in our correlation analysis. However, at 12 wks after the cessation
periosteal circumference (PC) was 18% ± 7.0 after the last load- of loading, we found that there was no difference in the yield
ing and was significant when compared with 7.2% ± 1.7 using load, maximum load and toughness between the loaded and
the higher threshold (730-730). This is due to rapid accumula- non-loaded bones (Table 1). This is because; the vBMD in the
tion of low mineralized bone at the periosteal surface. This is non-loaded bones increased with advancing age and the CSA
most prevalent immediately after the last day of loading, as seen decreased gradually in the loaded bones, together contributing
by the red color using the pQCT threshold indicator (Figure 4). for the loss of bone strength difference between the loaded and
Consequently, we found a significant increase in CSA immedi- non-loaded bones at 12 wks.
ately after the last day of loading, accompanied by an increase Some of the limitations of this study are as follows: 1) in
in vBMD. Over time, we found that the periosteal circumference the past, reports by others have predicted that some of the
in the loaded tibia tends to reduce gradually. As a result, the skeletal changes induced by four-point bending are due to pe-
magnitude of difference between loaded and non-loaded tibia riosteal pressure. In our previous QTL study, we found that
reduced at 12 wks after cessation of loading, resulting in no dif- sham-loading neither increased periosteal bone formation nor
ference in the periosteal circumference of loaded tibia between caused changes in expression levels of bone marker genes,
lower (PC, 9.13% ± 3.9) and higher threshold (PC, 9.8% ± 1.2). demonstrating that the skeletal changes induced by bending
These findings, suggest that the newly formed bone undergoes are not due to periosteal pressure16and this is also consistent
remodeling at the periosteal site to cortical bone over time which with earlier reports23. This finding also applies to the present
leads to a reduction in bone size in order to accommodate the study since we used the same loading model and regimen. 2)
loading induced changes in the bone shape. During this remod- In this study, we used 10 wk old mice for our four-point bend-
eling process, we found that the rate of loss in gained CSA (bone ing experiment. Since bones continue to grow beyond 10 wks
size) after cessation of loading was 10% every 2 wks for 8 wks. although at much lower pace this raises a question whether
These data, suggest that the positive effects of ML on CSA were growth has any effect on the response to loading. In the past,
278
C. Kesavan and S. Mohan: BMD fate after cessation of loading
we have performed four-point bending on three ages (10-, 16- in two breeds of mice. Calcif Tissue Int 1998;63(5):442-9.
and 36 wks) and reported that there was no significant differ- 3. Kesavan C, Mohan S, Oberholtzer S, Wergedal JE,
ence in the bone response between the age groups for a given Baylink DJ. Mechanical loading-induced gene expression
load. However, it remains to be determined whether the loss and BMD changes are different in two inbred mouse
rate in bone mass after cessation of loading is different in strains. J Appl Physiol 2005;99(5):1951-7.
young vs. old mice. 3) It is worth mentioning that the four- 4. Kodama Y, Umemura Y, Nagasawa S, et al. Exercise and
point bending method of loading also adds lamellar bone ini- mechanical loading increase periosteal bone formation
tially, however, as the duration of loading becomes larger, there and whole bone strength in C57BL/6J mice but not in
is an increase in accumulation of bone forming osteoblast cells C3H/Hej mice. Calcif Tissue Int 2000;66(4):298-306.
at the loading site. As a result, this gives rise to a large amount 5. Lang TF, Leblanc AD, Evans HJ, Lu Y. Adaptation of the
of low mineralized bone, so called woven bone over the lamel- proximal femur to skeletal reloading after long-duration
lar bone. Our study, however, cannot rule out the contribution spaceflight. J Bone Miner Res 2006;21(8):1224-30.
of woven bone vs. lamellar bone. 4) The four-point bending 6. Mori T, Okimoto N, Sakai A, et al. Climbing exercise in-
loading method applied in this study produced woven bone at creases bone mass and trabecular bone turnover through
the periosteal surface which is remodeled subsequently.  Since transient regulation of marrow osteogenic and osteoclas-
the site of loading is at the mid diaphysis which contains no togenic potentials in mice. J Bone Miner Res 2003;
trabecular bone, this method of loading is not applicable to 18(11):2002-9.
evaluate the loading response on lamellar bone.  Future studies 7. Turner CH, Robling AG. Exercise as an anabolic stimulus
using axial loading model which induces both trabecular and for bone. Curr Pharm Des 2004;10(21):2629-41.
cortical bone formation are needed to determine if axial load- 8. Bikle DD, Halloran BP. The response of bone to unload-
ing induces woven bone or lamellar bone at the trabecular site. ing. J Bone Miner Metab 1999;17(4):233-44.
Since exercise induces lamellar bone formation in humans, it 9. Bikle DD, Sakata T, Halloran BP. The impact of skeletal
is likely that axial loading in mice will also promote lamellar unloading on bone formation. Gravit Space Biol Bull
bone formation in the metaphysis of tibia. 5) In the past, we 2003;16(2):45-54.
have measured micro-cracks by histological analysis to rule 10. Dalsky GP, Stocke KS, Ehsani AA, Slatopolsky E, Lee
out the possibility whether the changes on bone parameters in- WC, Birge SJ, Jr. Weight-bearing exercise training and
duced by four-point bending are due to micro-crack induced lumbar bone mineral content in postmenopausal women.
healing. We found that there was no significant difference in Ann Intern Med 1988;108(6):824-8.
the micro-crack areas between loaded and non-loaded bones 11. Vuori I, Heinonen A, Sievanen H, Kannus P, Pasanen M,
of B6 mice. In the present study, we have used the same load- Oja P. Effects of unilateral strength training and detrain-
ing regimen and therefore, the response induced by four-point ing on bone mineral density and content in young women:
bending cannot be attributed to damaged induced bone forma- a study of mechanical loading and deloading on human
tion. Furthermore, we did not observe any fracture as evident bones. Calcif Tissue Int 1994;55(1):59-67.
from X-ray and pQCT image16. 12. Karlsson MK. The skeleton in a long-term perspective-are
In conclusion, our study shows that the positive effects of exercise induced benefits eroded by time? J Musculoskelet
Neuronal Interact 2003;3(4):348-51; discussion 356.
mechanical loading on bone were maintained for a substantial
13. Snow CM, Williams DP, LaRiviere J, Fuchs RK, Robin-
period of time after the cessation of the last external loading.
son TL. Bone gains and losses follow seasonal training
Acknowledgements and detraining in gymnasts. Calcif Tissue Int 2001;
69(1):7-12.
This work was supported by the Army Assistance Award No. DAMD17-
14. Iwamoto J, Yeh JK, Aloia JF. Effect of deconditioning on
01-1-744. The US Army Medical Research Acquisition Activity (Fort De-
cortical and cancellous bone growth in the exercise trained
trick, MD) 21702-5014 is the awarding and administering acquisition of-
young rats. J Bone Miner Res 2000;15(9):1842-9.
fice for the DAMD award. The information contained in this publication
15. Pajamaki I, Kannus P, Vuohelainen T, et al. The bone gain
does not necessarily reflect the position or the policy of the Government,
and no official endorsement should be inferred. All work was performed induced by exercise in puberty is not preserved through
in facilities provided by the Department of Veterans Affairs. We would a virtually life-long deconditioning: a randomized con-
like to thank Mr. James Dekeyser for his technical support of the four- trolled experimental study in male rats. J Bone Miner Res
point bending instrument and Peter Gifford for the animal work. 2003;18(3):544-52.
16. Kesavan C, Mohan S, Srivastava AK, et al. Identification
of genetic loci that regulate bone adaptive response to me-
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